Objective To explore the mechanism of eucalyptol, limonene and pinene enteric soft capsules in treating otitis media (OM) by regulating ferroptosis based on network pharmacology and molecular docking techniques, and to verify this mechanism through molecular dynamics simulation.

Methods The gene targets associated with eucalyptol, limonene and pinene enteric soft capsules and OM were obtained based on public databases, and the intersection of the two sets of targets were accquired. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) functional enrichment analyses were performed using Metascape database. A protein-protein interaction (PPI) network was constructed to screen key targets. Ferroptosis-related targets were retrieved from FerrDb database, and the intersection of eucalyptol, limonene and pinene enteric soft capsules, OM, and ferroptosis-related targets was selected. The core targets were screened using the CytoNCA plugin of Cytoscape 3.7.1 software and further enriched analysis was performed. Finally, molecular docking and molecular dynamics simulation were conducted to validate the interactions between eucalyptol, limonene and pinene enteric soft capsules and the ferroptosis-related targets.

Results 16 targets for eucalyptol, limonene and pinene enteric soft capsules in treating OM via regulating ferroptosis were screened, with the top 5 core targets being PPARG, HIF1A, MAPK3, PPARA, and MAPK1. The results of GO and KEGG enrichment analysis indicated that eucalyptol, limonene and pinene enteric soft capsules mainly treat OM by regulating cell differentiation, mitogen-activated protein (MAP) kinase activity, etc., and its regulation of ferroptosis depends on effercytosis, programmed death ligand-1/programmed death receptor-1 (PD-L1/PD-1), hypoxia-inducible factor-1 (HIF-1), and the central carbon metabolism of cancer. Molecular docking results demonstrated that three main components of eucalyptol, limonene and pinene enteric soft capsules exhibited binding energies below -5.0 kcal/mol with PPARG, HIF1A ,PPARA and MAPK1, among which eucalyptol-PPARA, limonene-MAPK3, and α-pinene-PPARG showed the best binding ability. The results of molecular dynamics simulation, including root mean square deviation (RMSD), energy, and root mean square fluctuation (RMSF), confirmed that the binding was highly stable.

Conclusion The active components of eucalyptol, limonene and pinene enteric soft capsules, including eucalyptol, limonene, and α-pinene, exert their therapeutic effects on OM by binding to core targets such as PPARA, MAPK3, and PPARG. These interactions regulate signaling pathways, including effercytosis, PD-L1/PD-1, HIF-1 and the central carbon metabolism of cancer, thereby providing a theoretical basis for the clinical application of eucalyptol, limonene and pinene enteric soft capsules in the treatment of OM.

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